Lipocortin 1 co-associates with cytokeratins 8 and 18 in A549 cells via the N-terminal domain.
An affinity chromatography strategy was used to search for proteins in A549 cells which interact with the N-terminus of lipocortin 1 (annexin 1). Using the biologically active fragment Lc13-25 as the affinity ligand, two proteins of molecular weight (m.w.) 52 and 48kDa were extracted. Affinity blots of these proteins bound iodinated Lc13-25. Partial tryptic digests of these proteins were analysed by matrix assisted laser desorption mass spectrometry and found to display fragmentation patterns with a strong similarity to those of cytokeratin 8 and 18 respectively. Subsequent blotting with a panel of specific cytokeratin antibodies strongly supported the idea that the two proteins were cytokeratin 8 and cytokeratin 18. Cytokeratin 8 was isolated from A549 cells in intermediate filament (IF) preparations which were also found to contain lipocortin 1 as a potential intermediate filament associated protein (IFAP). This association persisted throughout cycles of IF assembly and disassembly. Dual-labelling immuno-histochemistry in A549 cells showed strong co-localization of lipocortin 1 and cytokeratin 8. The implications of this finding are discussed in the light of the biological activity and possible function of lipocortin 1.
Croxtall
J D
JD
Department of Biochemical Pharmacology, The William Harvey Research Institute, The Medical College of St. Bartholomews and the Royal London School of Medicine and Dentistry at Queen Mary and Westfield College, London, UK. j.croxtall@qmw.ac.uk
Wu
H L
HL
Yang
H Y
HY
Smith
B
B
Sutton
C
C
Chang
B I
BI
Shi
G Y
GY
Flower
R
R
D017305:Annexin A1 / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry*; D007633:Keratins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D017421:Sequence Analysis
D017305:Annexin A1; D010446:Peptide Fragments; D007633:Keratins